Ribosomal frameshifting used in influenza A virus expression occurs within the sequence UCC_UUU_CGU and is in the +1 direction

A E Firth,J K Taubenberger,H M Wise,J F Atkins,C C Nelson,N M Wills,S Napthine,P Digard,K Parsawar,B W Jagger

doi:10.1098/rsob.120109

A E Firth, J K Taubenberger + Show 8 more

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https://doi.org/10.1098/rsob.120109

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Abstract

Programmed ribosomal frameshifting is used in the expression of many virus genes and some cellular genes. In eukaryotic systems, the most well-characterized mechanism involves –1 tandem tRNA slippage on an X_XXY_YYZ motif. By contrast, the mechanisms involved in programmed +1 (or −2) slippage are more varied and often poorly characterized. Recently, a novel gene, PA-X, was discovered in influenza A virus and found to be expressed via a shift to the +1 reading frame. Here, we identify, by mass spectrometric analysis, both the site (UCC_UUU_CGU) and direction (+1) of the frameshifting that is involved in PA-X expression. Related sites are identified in other virus genes that have previously been proposed to be expressed via +1 frameshifting. As these viruses infect insects (chronic bee paralysis virus), plants (fijiviruses and amalgamaviruses) and vertebrates (influenza A virus), such motifs may form a new class of +1 frameshift-inducing sequences that are active in diverse eukaryotes.

Highlights

During translation, shifts in reading register can occur to either alternative frame
The efficiency of frameshifting at the PA-X shift site was previously estimated by translating reporter constructs in rabbit reticulocyte lysates and found to be around 1.3 per cent [30]
Because PA-X is expressed at very low levels during virus infection, we were not able to isolate sufficient quantities from virus-infected cells for mass spectrometric analysis despite multiple attempts

Summary

Introduction

Shifts in reading register can occur to either alternative frame. In animals and fungi, such frameshifting is widely used to regulate expression of antizyme, the negative regulator of cellular polyamine levels [3,4] In both cases, protein sequencing has shown that the shift is þ1. The mammalian antizyme 1 frameshifting signals exclusively drive þ1 frameshifting in mammalian cells, they induce both þ1 and –2 frameshifting when a cassette containing them is expressed in Schizosaccharomyces pombe, and –2 frameshifting when expressed in Saccharomyces cerevisiae [5] In this system, the ratio of –2 to þ1 is alterable depending on the distance of a 30-adjacent stimulatory pseudoknot structure from the shift site [6]. Given similar sequences as in Ty1, the frameshifting used in decoding the mRNAs for actin filament binding protein ABP140 and telomere component EST3 is expected to be þ1 [15 –18]

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